Many diseases can be treated with a drug or a biologic agent (illustrative examples of biologic agents include nucleotides, e.g. siRNA, miRNA and the like; amino acids, including synthetic amino acids not occurring in nature; proteins, including enzymes, peptides, aptamers, antigens and the like; and antibodies, e.g. glycoproteins, immunoglobulins and the like). These drugs or biologics can be delivered into their target cells with targeting ligands, e.g. a folate receptor binding ligand, but their efficacy can be inhibited by an inability of the drug or biologic agent to be released from the endo some, for example, after folate-mediated endocytosis. Therefore discovery of new methods for “endosomal release” of trapped cargo into the cytoplasm would be useful for achieving increased efficacy of targeted drugs or biologics. It has been discovered that endosomal release can be facilitated by use of ligand ionophore conjugates to create osmotic pressure to rupture the endosomes containing the cargo using known ionophores that have low toxicity to healthy tissues. Without being bound by theory it is believed that nigericin, an ionophore and antiporter that couples efflux of H+ ions to influx of K+ ions, if delivered into cells, causes an osmotic imbalance inside endosomes leading to a swelling and/or disruption of the endosome and the release of the endosomal contents into cytoplasm. It will be appreciated that other K+ ionophores like salinomycin that transport potassium ions can also be employed for endosomal release.

In order to induce swelling of an endosome, an osmotically active ion can enter the endosome and promote the accompanying osmotically driven influx of water. This influx of water should force the endosome to enlarge, ultimately leading to its rupture. However, if the influx of the osmotically active ion is accompanied by the efflux of another osmotically active ion, no net change in water flow will occur and the endosome will not expand. Thus, for endosome swelling to occur, an osmotically active ion (e.g., Na+, K+, Li+, Ca++, Mg++) should enter the endosome in exchange for H+, which is the only osmotically inactive cation in nature. Moreover, because the only osmotically active ion that will flow spontaneously down its concentration gradient into an endosome is K+, an ionophore that is useful to lead to swelling of an endosome is an ionophore that can exchange K+ ions for H+ ions.
The Na+/H+ exchanger (antiporter) is a natural endosomal transporter whose function is to modify endosomal pH. It can work against a K+ ionophore-induced endosomal swelling by moving sodium ions out of the endosome in exchange for H+, leading to endosome shrinkage. Thus, the action of a K+ ionophore might be reduced by a naturally occurring Na+/H+ exchanger (antiporter), but augmented by the simultaneous addition of an inhibitor of the Na+/H+ exchanger such as amiloride, or HOE 694, or the like.
Folate receptors are over expressed on the cell membrane of many human cancers like ovarian, lung, breast, endometrium, brain, kidney and colon cancer and in activated macrophages which are responsible for inflammatory diseases like rheumatoid arthritis, artherosclerosis, osteoarthritis, diabetes, psoriasis etc. Folic acid has high binding affinity (Kd=10−10M) for folate receptors and can deliver releasable cargo to folate receptors in a selective manner avoiding off-site toxicity. Ligands bound to these receptors become part of the endosome that forms after the membrane invaginates into caveolae, internalizes and separates from the surface.
Prostate specific membrane antigen (PSMA) is a cell surface protein that is internalized in a process analogous to the endocytosis observed with cell surface receptors, such as folate receptors. It has been established that biologically active compounds that are conjugated via a linker to ligands capable of binding to PSMA may be useful in the imaging, diagnosis, and/or treatment of prostate cancer, and related diseases that involve pathogenic cell populations expressing or over-expressing PSMA. PSMA is over-expressed in malignant prostate tissues when compared to other organs in the human body such as kidney, proximal small intestine, and salivary glands. Although PSMA is expressed in brain, that expression is minimal, and most ligands of PSMA are polar and are not capable of penetrating the blood brain barrier. Unlike many other membrane-bound proteins, PSMA undergoes rapid internalization into the cell in a similar fashion to cell surface receptors like folate receptors. PSMA is internalized through clathrin-coated pits and subsequently can either recycle to the cell surface or be retained inside an endosome which progressively develops into a lysosome.
Even though a drug cargo delivered to a receptor capable of endocytosis, or an analogous process, is delivered selectively to the diseased cells, the path of delivered cargo to the cytoplasm or the nucleus can be blocked completely or partially by the invaginated plasma membrane called the ‘endosome’. Higher molecular weight agents, such as peptides, siRNAs, antisense oligonucleotides, proteins, aptamers, oligosaccarides and polysaccarides cannot escape endosomes once they have been internalized via a ligand-targeted endocytosis pathway. Thus the trapped cargo stays in the endosome and finally decomposes to smaller fragments by the action of acids and enzymes present in the endosome before being released in inactive form. The conjugates of the invention increase both the endosomal accumulation and escape of a therapeutic agent, or an imaging agent in targeted cells.
Several embodiments of the invention are described in the following clauses:
1. A conjugate comprising:
a ligand (B) targeted to a cell-surface receptor;
a linker (L); and
one or more ionophores (A) each of which couples efflux of protons (H+ ions) to influx of potassium ions (K+ ions);
wherein (L) comprises at least one releasable linker; (B) is covalently linked to (L); and each (A) is covalently linked to (L).
2. The conjugate of clause 1 wherein (L) comprises at least one releasable linker.
3. The conjugate of clause 1 or 2 further comprising a therapeutic agent, and/or an imaging agent wherein the therapeutic agent or the imaging agent is covalently linked to (L).
4. The conjugate of any of clauses 1 to 3 wherein (B) is targeted to a folate receptor or a prostate specific membrane antigen (PSMA).
5. The conjugate of clause 2 wherein (B) is a folate.
6. The conjugate of clause 5 further comprising a therapeutic agent.
7. The conjugate of clause 5 or 6 wherein (B) is folate.
8. The conjugate of clause 5 having the formula

9. The conjugate of clause 5 having the formula

10. The conjugate of clause 6 having the formula

11. The conjugate of any one of clauses 1 to 4 wherein (B) is a PSMA binding ligand;
12. The conjugate of clause 11 further comprising a therapeutic agent or an imaging agent.
13. The conjugate of clause 11 or 12 wherein the PSMA binding ligand is 2-[3-(1-carboxy-2-mercaptoethyl)ureido]pentanedioic acid (MUPA) or 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA).
14. The conjugate of clause 13 having the formula

15. The conjugate of any of the preceding clauses 3-4, 6-7 or 12-13 wherein the therapeutic agent comprises a low molecular weight drug, a polypeptide, a peptide, an oligonucleotide, a nucleotide, an siRNA, an iRNA, a microRNA, a ribozyme, an antisense oligonucleotide, a protein, a glycoprotein, an antibody, an antigen, a synthetic amino acid, an aptamer, an oligosaccaride, or a polysaccaride.
16. The conjugate of clause 15 wherein the therapeutic agent is siRNA, miRNA or iRNA.
17. The conjugate of clause 15 wherein the therapeutic agent comprises a low molecular weight drug.
18. The conjugate of clause 15 wherein the therapeutic agent comprises a peptide or a synthetic amino acid.
19. The conjugate of clause 15 wherein the therapeutic agent comprises a low molecular weight chemotherapeutic agent.
20. The conjugate of clause 19 wherein the therapeutic agent comprises a taxane or an analog thereof, a vinca alkaloid or an analog thereof, camptothecin or an analog thereof, a tubulysin or an analog thereof, or doxorubicin or an analog thereof.
21. The conjugate of clause 15 wherein the therapeutic agent comprises a low molecular weight anti-inflammatory agent.
22. The conjugate of clause 15 wherein the therapeutic agent comprises a lipophilic anti-inflammatory steroid.
23. The conjugate of clause 3 or 12 comprising an imaging agent.
24. The conjugate of clause 23 wherein the imaging agent comprises a fluorescent dye.
25. A conjugate of any of the preceding clauses wherein (A) is an inhibitor of the Na+/H+ exchanger.
26. The conjugate of clause 25 further comprising an ionophore wherein the ionophore couples efflux of protons (H+ ions) to influx of potassium ions (K+ ions).
27. The conjugate of clause 25 wherein the inhibitor is amiloride or HOE 694.
28. The conjugate of any of clauses 25-27 wherein the inhibitor is amiloride.
29. The conjugate of any of the preceding clauses 1-7, 11-13, 15-24 and 26-28 wherein the ionophore (A) is selected from the group consisting of nigericin or salinomycin.
30. The conjugate of clause 29 wherein the ionophore is nigericin.
31. The conjugate of any of clauses 1-7, 11-13 and 15-30 wherein (L) comprises a chain of about 7 to about 45 atoms.
32. A pharmaceutical composition comprising the conjugate of any of clauses 1-31, and 15-22 and further comprising at least one pharmaceutically acceptable carrier or excipient.
33. A pharmaceutical composition comprising the conjugate as described in any of clauses 3, 12 and 15-22 further comprising an additional therapeutic agent.
34. A method of increasing the endosomal accumulation and escape of a therapeutic agent, or an imaging agent comprising the step of administering with the therapeutic agent or the imaging agent an effective amount of a ligand-ionophore conjugate wherein the ionophore couples efflux of protons (H+ ions) to influx of potassium ions (K+ ions) and wherein the therapeutic agent or the imaging agent is targeted to a cell-surface receptor.
35. The method of clause 34 wherein the ionophore is selected from the group consisting of nigericin or salinomycin.
36. The method of clause 35 wherein the ionophore is nigericin.
37. The method of any of clauses 34-36 wherein the imaging agent or the therapeutic agent is not linked to the conjugate.
38. The method of any of clauses 34-36 wherein the imaging agent or the therapeutic agent is linked to the conjugate.
39. The method of clause 37 or 38 wherein the imaging agent or the therapeutic agent is targeted to the same receptor as the ligand-ionophore conjugate.
40. The method of clause 37 or 38 wherein the ligand-ionophore conjugate is the conjugate of any of clauses 1-2, 4 and 29-31.
41. The method of clause 39 wherein the ligand-ionophore conjugate is a conjugate of formula (B)-(L)-(A) and further comprises the imaging agent or the therapeutic agent, covalently linked to (L) and wherein the therapeutic agent or the imaging agent is as described in any of clauses 3 or 15-24.
42. The method of any of clauses 34-41 wherein the cell-surface receptor targeted by the ligand-ionophore conjugate is the folate receptor or the prostate specific membrane antigen (PSMA).
43. The method of clause 42 wherein the cell-surface receptor targeted by the ligand-ionophore conjugate is the folate receptor.
44. The method of clause 42 wherein the cell-surface receptor targeted by the ligand-ionophore conjugate is PSMA.
45. The method of clause 43 or 44 wherein the therapeutic agent or the imaging agent is targeted to a cancer or a site of inflammation.
46. The method of clause 45 wherein the cancer is selected from the group consisting of ovarian, lung, breast, prostate, endometrial, brain, kidney and colon cancer.
47. The method of clause 46 wherein the cancer is lung cancer.
48. The method of clause 46 wherein the cancer is ovarian cancer.
49. The method of clause 45 wherein the therapeutic agent or imaging agent is targeted to a site of inflammatory disease.
50. The method of clause 49 wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, atherosclerosis, diabetes, graft-versus-host disease, multiple sclerosis, osteomyelitis, psoriasis, Sjögren's syndrome, lupus erythematosus, Crohn's disease, and ulcerative colitis.
51. The method of clause 42 wherein the cell-surface receptor targeted by the ligand-ionophore conjugate is the prostate specific membrane antigen (PSMA).
52. The method of clause 51 wherein the ligand-ionophore conjugate is the conjugate described in any of clauses 11-24 and 29-31.
53. The method of clause 51 or 52 wherein the targeted cell-surface receptor is over-expressed PSMA.
54. The method of clause 53 wherein the therapeutic agent or the imaging agent is targeted to a malignant prostate cell population.
55. The method of any of clauses 34-54 comprising the administration of an inhibitor of the Na+/H+ exchanger (antiporter).
56. The method of clause 55 wherein the inhibitor of the Na+/H+ exchanger (antiporter) is amiloride or HOE 694.
57. The method of clause 55 or 56 wherein the inhibitor of the Na+/H+ exchanger (antiporter) is conjugated to the ligand.
58. The method of clause 55 or 56 wherein the inhibitor of the Na+/H+ exchanger (antiporter) is covalently linked to the ligand-ionophore conjugate and is releasable.
59. The method of any of clauses 34-58 wherein the imaging agent or the therapeutic agent is administered as a liposome, dendrimer or large molecular weight polymer complex in a targeted form.
60. The method of any of clauses 34-59 wherein the imaging agent or the therapeutic agent comprises an anticancer agent, an anti-inflammatory agent, a radionuclide, or a fluorescent dye.
61. The method of clause 60 wherein the therapeutic agent comprises a vinca alkaloid, doxorubicin, an antifolate or a corticosteroid.
62. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-20, 23-24 and 29-31 for the imaging or treatment of a cancer that expresses or overexpresses the folate receptor.
63. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-20, 23-24 and 29-31 for the manufacture of an agent for use in a method for imaging or treatment of a cancer that expresses or overexpresses the folate receptor.
64. An agent for use in imaging or treatment of a cancer that expresses or overexpresses the folate receptor, comprising a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-20, 23-24 and 29-31.
65. A method of using an effective amount of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-20, 23-24 and 29-31 in a method for imaging or treatment of a cancer, that expresses or overexpresses the folate receptor, in a subject in need thereof.
66. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 for imaging or treatment of an inflammatory disease at a site of inflammation.
67. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 for the manufacture of an agent for use in a method for imaging or treatment of an inflammatory disease at a site of inflammation.
68. An agent for use in imaging or treatment of an inflammatory disease, comprising a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31.
69. A method of using an effective amount of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 for imaging or treatment of an inflammatory disease in a subject in need thereof.
70. Use of a PSMA-targeting ligand-ionophore conjugate as described in any of clauses 11-14, 15-20, 23-24 and 29-31 for the imaging or treatment of a cancer that expresses or overexpresses PSMA.
71. Use of a PSMA-targeting ligand-ionophore conjugate as described in any of clauses 11-14, 15-20, 23-24 and 29-31 for the manufacture of an agent for use in a method for imaging or treatment of a cancer that expresses or overexpresses PSMA.
72. An agent for use in imaging or treatment of a cancer that expresses or overexpresses PSMA, comprising a folate-targeted ligand-ionophore conjugate as described in any of clauses 11-14, 15-20, 23-24 and 29-31.
73. A method of using an effective amount of a folate-targeted ligand-ionophore conjugate as described in any of clauses 12-13, 15-20, 23-24 and 29-31 in a method for imaging or treatment of a cancer, that expresses or overexpresses PSMA, in a subject in need thereof.
74. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 in association with a therapeutic agent or an imaging agent wherein the conjugate is internalized by endocytosis.
75. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 for the manufacture of an agent for use in a method for imaging or treatment of a cancer, for use in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis.
76. An agent for use in imaging or treatment of a cancer in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis, wherein the agent comprises a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31.
77. A method of using an effective amount of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 in a method for imaging or treatment of a cancer in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis.
78. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis for imaging or treating an inflammatory disease at a site of inflammation.
79. Use of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 for the manufacture of an agent for use in a method for imaging or treatment of an inflammatory disease in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis.
80. An agent for use in imaging or treatment of an inflammatory disease in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis, wherein the agent comprises a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31.
81. A method of using an effective amount of a folate-targeted ligand-ionophore conjugate as described in any of clauses 5-10, 15-18, 21-24 and 29-31 in a method for imaging or treatment of an inflammatory disease in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis.
82. Use of a PSMA-targeted ligand-ionophore conjugate as described in any of clauses 11-14, 15-20, 23-24 and 29-31 in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis, for the imaging or treatment of a cancer that expresses or overexpresses PSMA.
83. Use of a PSMA-targeted ligand-ionophore conjugate as described in any of clauses 11-14, 15-20, 23-24 and 29-31 for the manufacture of an agent for use in a method for imaging or treatment, in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis, of a cancer which expresses or overexpresses PSMA.
84. An agent for use in imaging or treatment of a cancer, in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis, wherein the agent comprises a PSMA-targeting ligand-ionophore conjugate as described in any of clauses 11-14, 15-20, 23-24 and 29-31.
85. A method of using an effective amount of a PSMA-targeting ligand-ionophore conjugate as described in any of clauses 11-14, 15-20, 23-24 and 29-31 in a method for imaging or treatment of a cancer that expresses or overexpresses PSMA, in association with a therapeutic agent, or an imaging agent that is internalized by endocytosis.